TSH receptor

ABSTRACT

A protein having the biological activity of a mammalian TSH receptor, and purified nucleic acid encoding such a protein.

This application is a divisional of U.S. Ser. No. 08/146,835, filed 29Oct. of 1993, now U.S. Pat. No. 5,614,363, which is acontinuation-in-part of U.S. Ser. No. 07/565,669, filed 10 Aug. of 1990,now abandoned, which is a divisional of U.S. Ser. No. 07/404,899, fled08 Sep. of 1989, now abandoned.

BACKGROUND OF THE INVENTION

This invention concerns nucleic acid encoding a mammalian thyroidstimulating hormone (TSH, also known as thyrotropin) receptor, andpurified mammalian TSH receptors.

The TSH receptor is a protein believed to be involved in a humanautoimmune disease termed "Graves'" disease. It is believed thatantibodies against the TSH receptor are made in patients suffering fromthis disease. These auto-antibodies are currently detected, by providingradiolabeled TSH, and detecting blocking of binding of the TSH to crudeporcine membranes thought to include a TSH receptor.

Rees Smith et al. (Endocrine Reviews 9:106, 1988) describes thestructure of a TSH receptor and predicts that clones of DNA encodingsuch receptors can be isolated by determination of the amino acidsequence of the TSH receptor and subsequent use of oligonucleotideprobes to identify clones in a library. The receptor was only purifiedto about 0.001% purity (i.e., 10 μg of TSH receptor in 1 g of protein.

SUMMARY OF THE INVENTION

Applicant has succeeded in isolating nucleic acid encoding at least twomammalian TSH receptors, and providing an expression system whichenables production of large amounts of purified mammalian TSH receptor.Such purified receptor is useful in detection of auto-antibodies inpatients suffering from Graves' disease or other malfunctions of thethyroid using simple antibody assays, such as a competitive radioimmuneassay or an ELISA test.

In a first aspect, the invention features purified nucleic acid encodinga protein having the immunological or biological activity of a mammalianTSH receptor. The purified nucleic acid can be purified cDNA, or apurified vector including that nucleic acid. In a related aspect, theinvention features purified, e.g., recombinant, protein having theimmunological or biological activity of a mammalian TSH receptor.

By "immunological activity" is meant the ability to selectively form animmune complex with auto-antibodies to the TSH receptor. By "purified"is meant that the nucleic acid or protein is provided separated fromcontaminating nucleic acid or other cell components, such as proteinsand carbohydrates, with which the naturally occurring nucleic acidencoding the receptor occurs. Most preferably, the nucleic acid isprovided as a homogeneous solution separated from all cell components,or is the major nucleic acid present in a preparation. More preferably,the nucleic acid is provided within a vector which is resident within acell in a manner which allows expression of the nucleic acid to providesufficient TSH receptor to be useful in this invention. By "recombinant"is meant that the protein is expressed from nucleic acid which has beenmanipulated by recombinant DNA methodology to place it in a vector orchromosome at a location in which it does not naturally occur.Preferably the purified protein is present at a purity of at least 10%of the total protein in a preparation, or even at 50% or 90% purity.

The biological activity of mammalian TSH receptor is that activitynaturally associated with the TSH receptor of mammals, i.e., the abilityof that protein to recognize and interact with TSH. It preferablyincludes other biological activities of the TSH receptor such asactivating adenylate cyclase, well known to those of ordinary skill inthe art.

In preferred embodiments the TSH receptor is that receptor occurring inhumans; the nucleic acid has a nucleotide sequence encoding an aminoacid sequence identical to that of a naturally occurring mammalian TSHreceptor, most preferably a human TSH receptor; or the nucleic acidencodes a protein having only conservative amino acid substitutionscompared to a naturally occurring mammalian TSH receptor. Suchconservative amino acid substitutions are well known to those skilled inthe art and would include, for example, substitution of valine forglycine or leucine, substitution of a positively charged amino acid foranother positively charged amino acid, or substitution of a negativelycharged amino acid for another negatively charged amino acid. Suchsubstitutions will not significantly affect the biological activity ofthe encoded TSH receptor; i.e., the biological activity of thesubstituted form will be at least 75% that of the naturally occurringform.

The proteins of the invention can be used in a method for detecting thepresence of anti-TSH receptor antibodies in the serum of a patient. Themethod includes providing a purified TSH receptor as described above,and contacting that receptor with the serum. Reaction of the receptorwith the serum is an indication of the presence of anti-TSH antibodiesin that serum. This method may include any of many well knownimmunological procedures for detection of antibodies, such as ELISA,Western blot or competitive binding assays.

The present invention provides a sufficient amount of a mammalian TSHreceptor to be useful for rapid testing of patients for the presence ofanti-TSH receptor antibodies. It also provides sufficient receptorprotein to allow analysis of the sequence of the protein. Such analysiswill aid determination of specific epitopes on that protein to allowdesign of small homologous peptides which will block the activity ofautoimmune antibodies. Those peptides will thus block overstimulation ofthe thyroid in patients, such as those suffering from Graves' disease.The invention also provides the tools necessary to allow development ofagonists or antagonists of TSH binding to a mammalian TSH receptor.These antagonists will be useful for preventing hyperthyroidism due toelevated levels of TSH.

In another aspect, the invention features a method for determining thepresence of TSH in a sample. The method includes providing a mammaliancell having DNA encoding biologically active TSH receptor, the cellexpressing TSH receptor from the DNA under assay conditions; contactingthe cell with the sample to cause TSH within the sample to contact thecell; and measuring the level of intracellular cyclic adenosinemonophosphate prior to and after the contacting step. An elevated levelof cyclic adenosine monophosphate after the contacting step compared toprior to the contacting step is indicative of the presence of TSH withinthe cell.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIG. 1 is a depiction of the nucleotide base sequence of the rat LHreceptor probe.

FIGS. 2A to 2E is a depiction of the nucleotide base sequence of thehuman LH receptor cDNA and the derived amino acid sequence.

FIG. 3 is a diagrammatic representation of the structure of the human LHreceptor-encoding gene and the derived amino acid sequence.

FIG. 4 is a depiction of the nucleotide base sequence of degenerateoligonucleotide probes based on the human LH receptor DNA sequence.

FIGS. 5A and 5B is a depiction of the partial nucleotide base sequenceof the bovine and human TSH receptors. The boxed sequences indicateregions with possible sequence errors due to compression during sequencedetermination.

FIGS. 6A to 6D is a depiction of the nucleotide base sequence and thederived amino acid sequence of the human TSH receptor cDNA.

FIG. 7 illustrates a darkfield photomicrograph (75×magnification)showing an autoradiographic signal (bright spots) produced byradiolabeled anti-sense transcript of human TSH receptor overlying ahaematoxylin and eosin stained section of human thyroid.

FIG. 8 is a diagrammatic representation of the pATH3-hTSHR expressionvector.

FIG. 9 illustrates a photograph of a polyacrylamide gel demonstratingthe expression of the trp E-TSH receptor fusion protein (small arrow) inthe absence (-) and presence (+) of indoleacetic acid. The small arrowindicates a protein of the size predicted for the fusion protein.

FIG. 10 is a graphical representation of the the level of intracellularcyclic adenosine monophosphate (cAMP) as a function of the concentrationof applied hormone.

TSH RECEPTOR

TSH receptors useful in this invention include any such receptorisolated from a mammal, or any protein having the biological activity ofsuch a receptor. Such proteins will include proteins derived fromnaturally occurring TSH receptors having one or more of their aminoacids modified conservatively as discussed above. Such modification maybe by any standard procedure, for example, by recombinant DNAtechnology. Generally,such receptors will be expressed by recombinantDNA technology by isolatingthe gene encoding that receptor, and placingthat gene within an expressionvector, after removing any intronic DNAthat may be deleterious to expression of the full length receptorprotein. Such expression vectors will include bacterial, fungal, insect,and mammalian expression vectors which may be expressed within abacterial, fungal, insect, or mammalian cell by techniques well known tothose with ordinary skill in the art. Purified mammalian TSH receptormay be also be isolated by preparing antibodies to one of the aboverecombinant mammalian TSH receptors, and using those antibodies toimmunoaffinity purify a naturally occurring TSH receptor. Generally,such a procedure is not preferred, since the yield ofTSH receptor willbe extremely small.

Once the desired TSH receptor protein is cloned, and its amino acidsequence determined, proteins having the biological activity of thereceptor may be designed by standard procedure. For example,oligonucleotides may be synthesized by standard procedure, and insertedinto any standard expression vector to cause expression of fragments ofthe naturally occurring TSH receptor. These fragments can be screened bystandard procedure to determine whether they have the desired biologicalactivity of the receptor protein. For example, it may be determined byaffinity chromatography, Western blot analysis, or some equivalentanalysis, whether that synthetic peptide is able to bind with antibodiesagainst a TSH receptor. Those fragments which can bind are useful inthis invention. Similarly, the expressed TSH receptor, or that purifiedas described above, may be fragmented by use of enzymes, e.g., trypsin,whichspecifically cleaves the amino acid sequence into smallerfragments. These fragments may then be tested in much the same way asthe synthetic peptidefragments to determine their usefulness in methodsof this invention.

Below is presented one example of a mammalian TSH receptor-encodinggene, and expression of that gene within a vector to provide a purifiedmammalian TSH receptor. This example is not limiting to the inventionand those skilled in the art will recognize many other mammalian TSHreceptorscan be isolated by identical procedures, or by use of thecloned DNA provided as deposits in the American Type Culture Collection(see below). The DNA in these deposits may be used to screen anyexisting or newly constructed library of mammalian DNA to determine thepresence of clones encoding a part or all of a mammalian TSH receptor.Preferably such libraries will be constructed as cDNA libraries from RNApresent in the thyroid of a mammal.

Example Human and Bovine TSH Receptor

A 622 nucleotide fragment of the rat luteinizing hormone (LH) receptorgenewas obtained from Deborah Segaloff of the Center for BiologicalResearch atthe Population Counsel, New York, N.Y., 10021, and from PeterSeeburg at the University of Heidelberg. This DNA fragment was used as aprobe of a lambda-gt11 cDNA library constructed from RNA isolated fromthe thyroid ofa patient suffering from Graves' disease. The nucleotidebase sequence of this probe is shown in FIG. 1.

The cDNA library was constructed generally as follows. RNA of thethyroid was isolated using a standard guanidium/thiocyanate procedureand reverse transcribed using the method of Gubler and Hoffman. Theresulting cDNA wassize selected using a Sepharose G50 gel filtrationcolumn to select cDNA ofgreater than 1 kb in size. The cDNA wasmethylated with EcoRI methylase, linked to EcoR1 linkers, and thentreated with EcoRI. The resulting DNA was ligated to EcoRI treatedlambda-gt11 DNA. The resulting lambda DNA wasamplified in E. coli strain1090.

The rat LH gene fragment was labeled with ³² P-dCTP and platescontaining the lambda gt11 library screened on nitrocellulose filters atlow stringency in 30% formamide, 1M NaCl, at 42° C. The filters werethen washed at low stringency in 2×SSC at 50° C.

Two classes of clones were detected, one class giving a strong reaction,and the other class a faint reaction with the probe. The stronglyreactingplaques were purified three times using standard procedure, andfour were determined to encode overlapping parts of the same gene byrestriction endonuclease mapping, and DNA sequencing procedures. The 5'terminal 600 nucleotides of the gene showed high homology to the rat LHreceptor. Further analysis determined that the cDNA encoded the fulllength human LHreceptor protein with several introns remaining. Thenucleotide base sequence is provided in FIG. 2. The amino acid sequence,molecular weight and isoelectric point of the encoded protein can becalculated by standardtechniques from this sequence. The encoded proteinhas 90% homology in amino acid sequence to the rat LH receptor protein.The cDNA includes intronic DNA. RNA protection experiments, Northernanalysis, and polymerase chain reaction experiments showed that the mRNAencoded by thisclone is expressed in the thyroid, testes, and ovary, aswell as in Graves'thyroid, and in thyroid cell lines. The RNA isexpressed in the thyroid butis incompletely spliced. Thus, this clonedoes not encode thyroid specific DNA.

In order to isolate clones encoding a TSH receptor, two degenerateoligonucleotide probes were constructed, one having homology to thetransmembrane domain III of the above cloned human LH receptor DNA andtheother having homology to the transmembrane domain VI of the LHreceptor DNA. These domains and the location of the probes are shown inFIG. 3. These domains are separated by a distance of approximately 400nucleotidesin the cDNA. The oligonucleotides were synthesized andpurified by standardprocedure; their sequences are shown in FIG. 4.

Total RNA was isolated from a human Graves' thyroid, and from a bovinethyroid sample. Ten μg of total RNA from these two samples wasseparately reversed-transcribed using Moloney murine leukemia virusreverse transcriptase (commercially available). First strand cDNA wassynthesized in a 50 μl reaction, and 5 μl of the resulting cDNA usedin apolymerase chain reaction with the above synthetic oligonucleotides.This reaction had a total volume of 100 μl , including 5 μl of cDNA,500picomoles of each oligonucleotide, and the standard buffers andnucleotides described by Cetus Corporation (Emeryville, Calif.). Thisreaction was treated at 94° C. for one minute in the presence of Taq DNApolymerase and then two minutes at 50° C. and three minutesat 72° C.This cycle of heating and cooling between 50° C. and 94° C. was repeatedthirty times. At this point, no amplification product could be observed.Five μl of the resulting reaction was removed and the procedurerepeated. At this point, a DNA product was observed. No such product wasobserved in reactions using total RNA isolated from osteosarcoma,testes, ovary, melanoma, or placenta. Thus, the DNA product appears tobe thyroid specific. The resulting material was precipitated andresuspended by standard procedure,and digested with HindIII and EcoRI.

The EcoRI HindIII fragment was subcloned into the vector pBS⁻(Strategene, La Jolla, Calif.), and transformed into E. coli. Theresulting vector was sequenced by Sanger dideoxy procedures. Both humanand bovine cDNAs were sequenced and found to encode a protein havingabout84% homology. Their tentative sequences are presented in FIGS. 5Aand 5B. In contrast, the DNA had only about 68% homology with rat,porcine, and human LH receptor.

The fragments derived from the polymerase chain reaction were removedfrom the vector and labeled with ³² P. These fragments were then used asprobes to screen the above described lambda-gt11 library at highstringency. The conditions were 50% formamide at 42° C. in the presenceof 1M NaCl for 15-20 hours, and then washing of the nitrocellulosefilters at 20°-25° C. in 2×SSC for 15, minutes at 68° C. in 1×SSC for 45minutes, and at 68° C. in 0.1×SSC for 45 minutes. Strongly hybridizingplaques were detected at a higher frequency than had been detected forthe LH receptor clones. Twelve of these plaques were purified threetimes, purified DNA isolated from six, and analyzed by EcoRI restrictionanalysis. Four of these clones contained inserts of approximately 4.2kb. These inserts wereinserted into the pBS⁻ vector.

Northern blot analysis using the resulting clones showed that the DNAhybridized to RNA expressed only in the thyroid in both Graves' patientsand the cold nodule sample, but not in the testes, ovary or othertissues.The DNA hybridized with an RNA of approximately 4.2 kb and thusappears to represent a full length clone of the human TSH receptor. ThisRNA has a 3'-untranslated sequence of between 2 and 2.5 kb, and a5'-untranslated sequence of approximately 50 bases. One clone, TR.12.6-1(hTSH receptor), has been determined, by DNA sequencing, to contain afull length human TSHreceptor cDNA (FIGS. 6A to 6D).

Further proof that the clone encoded human TSH receptor was provided byin situ hybridization histochemistry which demonstrated specifichybridization of anti-sense human TSH receptor probe to thyroidfollicularcells which are known to respond to TSH (FIG. 7). Briefly, 8μm cryostatsections of normal appearing thyroid follicles were preparedfor in situ hybridization. A 1 kb fragment of cDNA encoding the humanTSH receptor wasused to prepared ³⁵ S labelled anti-sense transcript.Tissue sections were pre-treated with detergent and protease, and thenincubated in hybridization buffer for 16 hours at 42° C. with 3×10⁵ CPM(specific activity approximately 10⁸ cpm/μg) of probe as described(Hoefler et al., Histochem, J. 18:5597, 1986).

The above-described cDNA from human and bovine, or any other mammal maybe expressed by standard procedures to provide large quantities of TSHreceptor. For example, the above cDNA may be inserted into a trpE-fusion plasmid, e.g., pATH-1, 2, or 3, to form a stable hybrid proteinwith the Trp E protein. Alternatively, the cDNA may be inserted into amammalian expression system such as a cytomegalovirus or retrovirusvector. Glycosylated protein will result when the DNA is expressed inthe mammalian expression system.

Below is presented an example of a method to express TSH receptor. Theamino terminal coding sequence of the human TSH receptor from a PstIsite (nucleotide 346) to a HindIII site (nucleotide 1213) was ligated toPstI/HindIII digested pATH3. The resulting plasmid, pATH3-HTSHR (FIG.8), expresses a 66 kD fusion protein containing approximately 37 kD ofthe E. coli Trp E protein fused to 29 kD of the amino terminus of humanTSH receptor protein. DH5αE. coli transformed with pATH3-hTSHR weregrown in selective M9 media for 2 hours in either the absence orpresence of 40 μg/μl indoleacetic acid, an inducer of the Trp E gene.Bacterial pellets were lysed in SDS loading buffer and 1/10 th of thematerial was electrophoresed on a 10% polyacrylamide Laemmeli gel (FIG.9).

Use

As discussed above, nucleic acid encoding TSH receptor may be used toexpress large quantities of TSH receptor. For example, high levelexpression is achieved with a Baculovirus vector pVL941, the E. colivector paTH3, and the mammalian vector pLJ. Such protein is useful fordetection of auto-antibodies found in Graves' patients. This allowsdetermination of the state of the thyroid of those patients, andindicatesthe progress of that patient. This test may be performed in anELISA format, for example, in a dipstick assay. The test might also takethe form of a competitive binding assay employing radiolabeled TSH andTSH receptor. Such assays are extremely sensitive, and more readilyperformed than prior methods of detecting such antibodies.

The expressed protein is useful for defining the epitopes recognized byantibodies in Graves' patients. This analysis may be performed bystandardprocedure, for example, by expressing portions of the cloned DNAto providepartial TSH receptor fragments, or by fragmenting theexpressed receptor protein as discussed above. Once the regionrecognized by such antibodies is defined, these fragments may be used inimmunoassay procedures. In addition, definition of epitopes may beperformed by manipulating the cloned genes using standard techniques ofmolecular biology to provide proteins in which one or more amino acidswhich may form a part of one or more epitopes of the protein is alteredor deleted.

The protein or portions thereof is also useful as a therapeutic where itmay be administered in a pharmaceutically acceptable compound at asufficient dose to alleviate one or more symptoms of Grave's patients,or other patients suffering from thyroid malfunction. Generally, suchadministration will be at a level between one and one thousandmicrograms per kilogram of patient.

Small peptides may be designed which will block the activity ofauto-antibodies that act as TSH agonists, and thus block stimulation ofthe thyroid. Other small peptides may be designed which will blockauto-antibodies that act as TSH antagonists. In addition, antagonists ofTSH may be constructed which prevent binding of TSH to the TSH receptorand thus prevent elevated thyroid activity.

Assays for TSH

There follows two assays for TSH. The first assay technique is basedupon the expression of TSH receptor within a cell which does notnaturally contains such a receptor. This cell, when contacted with TSH,will increase expression of cyclic adenosine monophosphate, which can bedetected as a measure of the amount of TSH in a sample.

In this assay, the human TSH receptor-encoding DNA is inserted with amammalian retroviral vector pLJ at the BamHI to SalI sites. Theresulting vector is then transfected into human 293 cells and clonalcell lines containing the vector isolated by selection in the presenceof the antibiotic G418. Such transfection causes the cells to becomeresponsive to TSH as measured by the activation of adenylate cyclase andaccumulationof cAMP following treatment with TSH. Thus, these cell linesprovide a highly sensitive assay system for the hormone TSH. Cells inculture or cell membrane preparations may be exposed to the samplethought to containTSH and the resulting adenylate cyclase activityquantitated and correlatedwith the cyclase activity from standarddilution curves of TSH in order to calculate the concentration of TSH ina sample. Concentrations as low as 1ng/ml or even 0.1 ng/ml can bedetected in this assay. This assay demonstrates that the TSH receptorencoded by the cDNA described above is biologically active and leads tospecific TSH responsiveness in a previously unresponsive cell line.These cell lines are responsive not only to naturally occurring TSH butalso to recombinant TSH.

Specifically, a retrovirus expression vector pLJ (Korman et al., Proc.Natl. Acad. Sci. USA 84:2150, 1987) containing the entire tr.12 cDNAsequence was transfected into human 293 cells and intracellular cAMPconcentrations measured 60 hours later using a ³ H-cAMP displacementassay after treatment with hCG, hFSH, or hTSH. Referring to FIG. 10, 100ng/ml of hFSH or hCG has little effect while the same amount of hTSHelevated intracellular cAMP over 6-fold. Half maximal intracellularconcentrations of cAMP were obtained with approximately 60 picomolarhTSH.In several experiments, a 15-fold elevation of intracellular cAMPwas induced by application of 100 ng/ml hTSH. Transfection of theretrovirus vector alone, with no hTSH-r insert, produced no elevation ofintracellular cAMP over background in cells treated with 100 ng/ml TSH.Expression of the human LH/CG receptor was attempted using identicalmethods, however, no elevation of cAMP was seen after treatment with anyof the glycoprotein hormones. This could result from any of number ofproblems, including, for example, the deletion found in clone tr.13, orperhaps inefficient removal of the LH/CG-R introns in the non-gonadal293 cell line.

The TSH receptor of this invention can be used to measure TSH by meansof acompetitive binding assay. In this assay TSH receptor, or a portionthereofcapable of binding TSH, is immobilized on a support matrix. Theimmobilizedreceptor is incubated with excess TSH, which has been taggedwith a radioactive or florescent label, long enough for the bindingreaction to come to equilibrium. Unbound TSH is removed by a washingstep, and the receptor is incubated with the test sample. Once thissecond binding step has come to equilibrium, the immobilized receptor iswashed again. The amount of tagged TSH displace by TSH in the testsample then serves as a measure of the TSH present in the test sample.Other assays for TSH employing purified TSH receptor can be devised bythose skilled in the art.

Deposits

The following DNA deposits were made on Sep. 6, 1989, with the AmericanType Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md.28052 under the terms of the Budapest Treaty, where the deposits weregiven the following accession numbers:

    ______________________________________                                        Deposit          Accession No.                                                ______________________________________                                        tr.12.6-1 (hTSH receptor)                                                                      40651                                                        tr.13.t35 (hLH receptor)                                                                       40652                                                        ______________________________________                                    

Applicant's assignee, New England Medical Center Hospitals, Inc.,represents that the ATCC is a depository affording permanence of thedeposit and ready accessibility thereto by the public if a patent isgranted. All restrictions on the availability to the public of thematerial so deposited will be irrevocably removed upon the granting of apatent. The material will be available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and35 USC 122. The deposited material will bemaintainedwith all the care necessary to keep it viable anduncontaminated for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited microorganism,and in any case, for a period of at least thirty (30) years after thedate of deposit or for the enforceable life of the patent, whicheverperiod is longer. Applicants' assignee acknowledges its duty to replacethe deposit should the depository be unable to furnish a sample whenrequested due to the condition of the deposit.

Other embodiments are within the following claims.

I claim:
 1. A purified nucleic acid comprising the nucleotide sequenceset forth in FIG. 6 or a nucleotide sequence complementary to that setforth in FIG.
 6. 2. A vector comprising a nucleic acid of claim
 1. 3. Acell into which a nucleic acid of claim 1 has been introduced.
 4. A cellinto which a vector of claim 2 has been introduced.
 5. A purifiednucleic acid which hybridizes under stringent conditions to a nucleicacid of claim
 1. 6. A vector comprising a nucleic acid of claim
 5. 7. Acell into which a nucleic acid of claim 5 has been introduced.
 8. A cellinto which a vector of claim 6 has been introduced.
 9. A purifiednucleic acid of claim 5, wherein said stringent conditions compriseincubation in 50% formamide at 42° C. in the presence of 1M NaCl for15-20 hours, followed by washing at 20°-25° C. in 2×SSC for 15 minutes,followed by washing at 68° C. in 1×SSC for 45 minutes, followed bywashing at 68° C. in 0.1×SSC for 45 minutes.
 10. A nucleic acid of claim5, wherein said nucleic acid is cDNA.
 11. A purified nucleic acidencoding a thyroid stimulating hormone (TSH) receptor protein having theamine acid sequence set forth in FIG.
 6. 12. A vector comprising anucleic acid of claim
 11. 13. A cell into which a nucleic acid of claim11 has been introduced.
 14. A cell into which a vector of claim 12 hasbeen introduced.
 15. A nucleic acid of claim 2, wherein said nucleicacid is cDNA.